Zero length conformation-dependent cross-linking of phosphorylase kinase subunits by transglutaminase.

Transglutaminase, a zero length cross-linker that catalyzes the formation of isopeptide bonds between proximal Gln and Lys side chains, was used as a structural and conformational probe of the hexadecameric phosphorylase kinase molecule (alpha beta gamma delta)4. Brief cross-linking of nonactivated kinase caused formation of alpha-beta dimers, with no cross-linking involving the gamma- and delta-subunits. When the kinase was first activated by autophosphorylation, significant amounts of alpha-alpha dimers were also observed in addition to the alpha-beta, demonstrating the occurrence of a conformational change in the alpha-subunits concomitant with activation. Both dimers resulted from intramolecular cross-linking. Because the COOH-terminal regions of the alpha-subunits are at the lobe tips of this bilobal kinase (Wilkinson D. A. Marion, T. N., Tillman, D. M., Norcum, M. T., Hainfeld, J. F., Seyer, J. M., and Carlson, G. M. (1994) J. Mol. Biol. 235, 974-982), the formation of zero length cross-linked alpha-alpha dimers indicates that the polypeptide backbones of these subunits must stretch from the lobe tips to a more central location where they abut each other. Excess putrescine, as the amine substrate in place of endogenous Lys, was incorporated by transglutaminase predominately into the alpha-subunits of the kinase, with only slight modification of the beta- and gamma-subunits. Exogenous calmodulin (delta'), an activator of the kinase with a binding site on the alpha-subunits (James, P., Cohen, P., and Carafoli, E. (1991) J. Biol. Chem. 266, 7087-7091), was a potent inhibitor of cross-linking. It also inhibited incorporation of putrescine into the alpha-subunits but stimulated incorporation into the beta- and gamma-subunits. Heparin, another activator of the kinase, had the same effects as exogenous calmodulin on cross-linking and putrescine incorporation, suggesting a commonality in the mechanism through which these two effectors activate the holoenzyme, including promoting a conformational change that increases the surface accessibility of target Gln residues on the catalytic gamma-subunit.